MULTINUCLEATED GIANT CELL FORMATION AND PHENOTYPE

Multinucleated giant cells (MGC) are homotypic macrophage syncytia associated with granulomas. Despite their correlation with pathology, MGC functional contributions to inflammation are relatively unknown. The objective of this work was to gain an understanding of MGC phenotype. First, techniques were developed to better enable the study of these cells in vitro. Second, inorganic particles known to cause inflammation were observed to cause MGC formation in the lungs. Finally, the particle that resulted in the highest macrophage fusion was used together with the in vitro system to compare MGC and macrophage phenotype in response to stimulation. The results contribute to fundamental MGC cell biology knowledge that is important toward developing approaches to control the foreign body response and understanding the role of MGC in granulomatous disease

Carbon Nanotubes for Bone Tissue Engineering

Biological tissues are compositionally and structurally exquisite – a complex network of proteins and cells organised with molecular-precision. Unfortunately, in the absence of an organ transplant or tissue graft, there are no technologies that can completely repair or restore this complex system when it fails. With the hopes of regenerating failing tissue, tissue engineers have developed scaffold structures able to support cell life. As yet, these structures are unable to recreate the complexities of the biological environment, limiting the success of this approach. Nanotechnologies have realised methods to make materials with defined nanoscale properties. Continued research may lead to sophisticated nanobiomaterials, with properties that rival the complexities of biological environments and improve tissue regeneration. To this end, we explored the use of carbon nanotubes (CNTs) within the field of tissue engineering. We investigated the use of 3D CNT scaffolds in bone tissue engineering using strong and porous ceramic scaffold structures coated with CNTs. We abate limitations in previous fabrication methods limiting coating of CNTs throughout porous structures. We demonstrate these surfaces are high quality aligned CNTs, are non toxic and able to support attachment, spreading and proliferation of adipose derived stem cells (ASCs) and human osteoblasts. Following the development of a 3D CNT material, we investigated the potential for using CNTs to create well-defined nanoenvironments capable of regulating cell differentiation. This research is the first report of non-biased quantitative measurement of cell shape during long term differentiation. In contrast to previous techniques, it allows direct measurement of shape rather than that of the underlying substrate. This approach offers novel insights into the relationship between the nanoenvironment, cell shape and cell differentiation. The novel nanomaterials presented in this thesis, demonstrate the potential of nanotechnologies for artificially engineered tissues and organs. Continued research of nanomaterials promises to better recreate the complexities of the biological environment, instructing healthy regenerative processes and promoting tissue function

The role of nanomaterial-protein interactions in determining the toxic consequences of nanomaterial exposure

As early biological responses to foreign objects in the body can be influenced by their bound proteins, the nanomaterial hard protein corona (the collection of slow exchange proteins that associate with nanomaterials) is an emerging area of interest in nanotoxicology. There is a limited but growing appreciation of the role these interactions have in influencing nanomaterial toxicity. This research dealt with (i) the characterisation of iron oxide and silica particles with and without a plasma, serum and lung lining fluid protein hard corona, (ii) the identification of the proteins in the hard corona that associate with the particles and (iii) the effect of the hard corona on influencing particle cytotoxicity in a J774.A1 macrophage cell line. Initial investigation of the particles illustrates the advantages in using a variety of characterisation techniques to better elucidate particle properties. Subsequent characterisation of the hard corona protein profile demonstrated a clear difference in the biological identity of the particles in a plasma, serum and in a lung lining fluid corona. Although it is difficult to associate the impact of any individual protein identified in the hard corona to cytotoxicity this study indicates that the binding of proteins plays a significant role in altering the cytotoxic potential (as determined by LDH release) in macrophages. The work also demonstrates the hard corona has an impact on macrophage chemotaxis, which further strengthens the hypothesis that the corona is a key consideration in nanoparticle toxicity. Ultimately this thesis finds that the nanomaterial hard corona is an important element to consider in experimental design and highlights the concept of creating particle preparation protocols to mimic the corona composition in vivo when examining in vitro cellular responses. This research highlights the implications for interpretation of data from in vitro cell culture tests that do not take the protein corona into consideration

Characterization of interaction of magnetic nanoparticles with breast cancer cells

Background: Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells. Results: Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells. Conclusions: All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cellsThe research leading to these results have received partial funding from the European Seventh Framework Programme (FP7/2007-2013) under the project MULTIFUN grant agreement no. 262943, and the project Nanofrontmag-CM (S2013/MIT-2850) from the Comunidad de Madrid. Additional grants were obtained from BFU 2011–29038 and CTQ2013-48767-C3-3-R from the Ministerio de Economia y Competitividad and S2009/Mat 1507 from the Comunidad de Madrid (to JLC), from EU FP7 project NAMDIATREAM (ref 246479) and from “la Caixa” / CNB International PhD Programme Fellowship

A multimodal approach to identify asbestos and characterise clinically relevant mesothelioma biomarkers

Malignant mesothelioma (MM) is an aggressive cancer of the mesothelium, with long latency and poor overall survival (OS), associated with occupational and environmental exposure to asbestos and other mineral fibres (MF). Considering that early diagnosis has been linked to an improvement in prognosis and treatment response, there is an urgent need for reliable MM biomarkers. Additionally, current asbestos identification techniques lack sensitivity and fail to detect shorter fibres or fibre fragments. Accurately identifying MF within MM samples is not only essential in aiding early diagnosis, but it also plays a key role in linking this diagnosis to asbestos exposure, which has high implications in legal, social, and political matters. The aim of the current project is to (1) develop MM cellular models suitable for laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging, (2) identify various MF based on their metal content within MM in vitro models as well as patient samples using a combination of LA-ICP-MS and LA-ICP-time of flight (TOF)MS instrumentation, (3) investigate the MM metallome in human tissue samples using LA-ICP-MS elemental mapping, and (4) characterise a panel of emerging MM biomarkers using a multi-modal approach. For the first time, this study has developed an asbestos detection technique using LA-ICP-MS imaging to identify MF within MM samples. High-resolution and high-speed analysis suggests that LA-ICP-MS imaging has the potential to be ultimately integrated in the clinical workflow to aid the identification of patients at an earlier and more treatable stage to improve survival outcomes. Moreover, the selected panel of biomarkers was characterised in cellular models, whilst novel biomarkers were tentatively identified using matrix assisted laser desorption ionisation (MALDI)-MS imaging of tissue microarrays, setting the basis for vital future investigations, and ultimately supporting MM biomarker discovery, early diagnosis, and improved prognosis

OXIDATIVE LUNG INJURY AND ITS PERSISTENT MITOGENOMIC EFFECTS IN HUMAN AIRWAY EPITHELIAL CELLS

The mitochondrial genome is a small, plasmid-like chromosome which encodes only 13 protein subunits yet is vital for electron transport in the mitochondrion and, therefore, vital for the existence of multicellular life. Despite this importance, mitochondrial DNA (mtDNA) is found in one of the least-protected areas of the cell, and is exposed to high concentrations of intracellular reactive oxygen species (ROS) and threat from exogenous substances and pathogens. Until recently, the quality control mechanisms that ensured the stability of the nuclear genome were thought to be minimal in the mitochondria. However, a vast network of mechanisms has been discovered that repair mtDNA lesions, replace and recycle mitochondrial chromosomes, and conduct alternate RNA processing for previously undescribed mitochondrial proteins. New mtDNA/RNA-dependent signaling pathways reveal a mostly undiscovered biochemical landscape in which the mitochondria interface with their host cells/organisms. As the myriad ways in which the function of the mitochondrial genome can affect human health have become increasingly apparent, the use of mitogenomic biomarkers (such as copy number and heteroplasmy) as toxicological endpoints has become more widely accepted. The objective of this dissertation was to investigate the role of mitochondrial genotoxicity in exacerbation and susceptibility to inflammation of the human airway epithelium. To do this, primary bronchial epithelial cells (BECs) were collected from human donors, healthy and those with chronic inflammatory lung diseases, and grown through multiple passages in serum-free culture. Disease groups were compared to healthy cells for mitogenomic markers, including expression of mtDNA genes, ultradeep sequencing for heteroplasmic frequency and mutant alleles, mtDNA lesions, and processing of polycistronic mtRNA using Nanostring probes. These results were compared to functional endpoints, including oxygen consumption rate, mitophagy, and cell viability/apoptosis, to determine whether observed mitogenomic stress had effects on epithelial cell function. The results of these studies indicated that severe chronic lung inflammation associated with fatal asthma and cystic fibrosis can cause impaired expression of mitochondrial genes, perhaps due to failed mtRNA processing. Similar endpoints were also examined following in vitro exposure of healthy BECs to nanomaterials, to examine acute genotoxic effects on mtDNA. Nanoparticle exposures had little mitogenomic effect, though carbon nanotubes did significantly decrease mtDNA expression, secondary to induction of mitophagy and decreased mitochondrial abundance.Doctor of Philosoph

Proteomic Characterization of Biological Effects Induced by Engineered Nanomaterials

Nanotechnology has been one of the major success stories of the early 21st century. The foundation for this success rests on the discovery that a small size confers completely new properties on materials. Nowadays, engineered nanomaterials (ENMs) are used in a plethora of applications such as paints, cosmetics, food products and electronics. These new properties, however, potentially make ENMs more reactive in biological systems than their large-scale counterparts. Already, asbestos-like effects have been described in mice after exposure to certain forms of carbon nanotubes (CNTs), while nano-sized titanium dioxide (nTiO2) has been shown to evoke inflammation in mouse lung. Therefore, extensive nanosafety studies have to be performed to ensure that no adverse effects are suffered by either workers or end-users of ENM products. This thesis has investigated the biological effects of ENMs by proteomic methods, first by evaluating the uptake and interactions of ENMs with plasma and cellular proteins followed by an analysis of the effects of ENM exposure on the intracellular proteome and secretome of human primary macrophages. The results revealed that ENM interactions with cellular proteins were governed by the surface reactivity of ENMs, whereas interactions with plasma proteins seemed to depend on the combination of both surface reactivity and active recognition, namely tagging of ENMs by opsonin proteins. The binding of cellular proteins to ENMs and subsequent interference with cellular processes might represent a novel cause of ENM toxicity, especially since transmission electron microscopy (TEM) micrographs indicated that several ENM species could be visualized free in the cytoplasm. The cytoplasmic protein expression changes after exposure to silica coated and uncoated nTiO2 revealed that silica coated TiO2 induced stronger protein expression changes in the macrophages. Most of the proteins with altered expression were related to phagocytosis, oxidative stress and inflammation. These proteome changes indicate that macrophages are actively engulfing ENMs and processing them. Moreover, the up-regulation of oxidative stress related proteins might be an indication of oxidative burst. Finally, nTiO2 treatment evoked acetylation of cytoplasmic proteins, a previously uncharacterized phenomenon in cells exposed to ENMs. The results from the macrophage secretome analysis showed that asbestos and long rigid carbon nanotubes (R CNTs) produced a similar response, while protein secretion profile of macrophages exposed to long tangled carbon nanotubes (T CNTs) exhibited a distinct profile. Bioinformatic analysis revealed that R CNTs evoked secretion of inflammation and apoptosis related proteins, possibly because of lysosomal damage. Functional assay confirmed that R CNT exposure triggered apoptosis in macrophages, while T CNTs and asbestos did not. This thesis offers new knowledge concerning the biological effects of engineered nanomaterials. Proteomic methods proved to be useful in the ENM-protein interaction studies revealing that it would be beneficial to include the ENM-protein interaction experiments as part of the routine ENM characterization when assessing the health effects of ENMs. By employing quantitative proteomics, we obtained a global view of both cytoplasmic and secreted proteome changes of macrophages exposed to different ENMs.Nanoteknologia on ollut 2000-luvun alun suurimpia menestystarinoita, joka sai alkunsa kun keksittiin, että pieni koko antaa materiaaleille kokonaan uudenlaisia ominaisuuksia. Nykyään teollisia nanomateriaaleja (TNM) käytetään lukuisissa sovelluksissa, kuten maaleissa, kosmetiikassa, elintarvikkeissa ja elektroniikassa. Pieni koko saattaa kuitenkin aiheuttaa myös ongelmia, sillä nanokokoiset materiaalit saattavat reagoida biologisissa ympäristöissä eri tavalla kuin saman aineen suurempi muoto. Eräiden hiilinanoputkimuotojen onkin jo havaittu aiheuttavan asbestinkaltaisia vaikutuksia hiirimalleissa, lisäksi nanokokoisen titaanidioksidin (nTiO(2)) on raportoitu aiheuttavan keuhkotulehdusta hiirikokeissa. Muun muassa näistä syistä, teollisten nanomateriaalien turvallisuutta olisi tutkittava laajasti jotta vältytään terveysriskeiltä, joille ovat alttiina sekä nanoteollisuudessa työskentelevät että kuluttajat. Tässä väitöskirjassa on tarkasteltu teollisten nanomateriaalien biologisia vaikutuksia proteomiikan menetelmillä. Ensimmäisessä osatyössä tutkittiin TNM:ien pääsyä soluihin ja vuorovaikutuksia proteiinien kanssa. Toisessa osatyössä analysoitiin TNM altistuksen aiheuttamia muutoksia makrofagien soluliman proteomissa ja viimeisessä tarkasteltiin TNM altistuksen aikaansaamia vaikutuksia makrofagien erittämiin proteiineihin. Tulokset osoittivat että tärkein teollisten nanomateriaalien ja soluproteiinien vuorovaikutuksia määrittävä tekijä oli TNM:ien pinnan reaktiivisuus. Sitä vastoin TNM:ien ja plasmaproteiinien vuorovaikutukset perustuivat sekä materiaalien pinnan reaktiivisuuteen että tiettyjen plasmaproteiinien, kuten opsonisaatiossa toimivien proteiinien, kykyyn tunnistaa aktiivisesti vieraita molekyylejä. Teollisten nanomateriaalien tarttuminen solun proteiineihin voi mahdollisesti aiheuttaa toksisia reaktioita häiritsemällä proteiinien toimintaa, varsinkin kun läpäisyelektronimikroskooppikuvissa useat eri nanomateriaalimuodot näyttivät päässeen ulos kalvorakenteiden sisältä solulimaan. Vertailtaessa silikalla päällystetyn nTiO(2):n ja päällystämättömän nTiO(2):n vaikutuksia makrofageissa, voitiin havaita että silikalla päällystetty nTiO(2) aiheutti voimakkaamman muutoksen soluliman proteomissa kuin päällystämätön. Makrofageissa tapahtuvat proteomimuutokset viittasivat aktiiviseen nTiO(2) hiukkasten fagosytoosiin eli solusyöntiin, makrofageissa tapahtuvaan happirasitukseen sekä tulehdusreaktioihin. Happirasituksessa aktivoituvien proteiinien määrän kasvu kertoi mahdollisesti happiradikaalien määrän hetkellisestä räjähdysmäisestä kasvusta (oxidative burst), joka on yleinen makrofageissa tapahtuva reaktio fagosytoosin jälkeen. nTiO(2) altistus aiheutti myös solunsisäisten proteiinien asetyloitumista, jota ei ole aikaisemmin havaittu nanomateriaaleilla altistetuissa soluissa. Kun verrattiin asbestin, pitkien ja jäykkien hiilinanoputkien (R CNT) sekä pitkien ja lankakerämäisten hiilinanoputkien (T CNT) aiheuttamia muutoksia makrofagien erittämien proteiinien määrään ja laatuun, voitiin havaita että R CNT ja asbesti aiheuttivat samankaltaisen reaktion makrofageissa. T CNT:n aiheuttamat muutokset makrofagien erittämissä proteiineissa sen sijaan olivat erilaisia. Bioinformatiikka-analyysit osoittivat että R CNT:lla altistetut makrofagit erittivät tulehdukseen ja solukuolemaan liittyviä proteiineja, mikä mahdollisesti johtui lysosomien hajoamisesta. Lisäkokeilla pystyttiin osoittamaan, että R CNT oli ainoa testatuista materiaaleista joka sai aikaan merkittävää solukuolemaa makrofageissa. Tämä väitöskirja on tuottanut uutta tietoa teollisten nanomateriaalien biologisista vaikutuksista. Käytetyt proteomiikan menetelmät osoittautuivat toimiviksi tutkittaessa TNM:ien ja proteiinien vuorovaikutuksia. Koska teollisten nanomateriaalien pinnan reaktiivisuus vaikuttaa myös materiaalien toksisuuteen, nämä vuorovaikutuskokeet voisivat olla osa koepaneelia, kun selvitetään TNM:ien terveysvaikutuksia. Proteomiikan avulla saatiin myös kokonaisvaltainen näkökulma TNM:ien vaikutuksista makrofagien sisäiseen proteomiin sekä proteiinieritykseen

MECHANISMS AND CONSIQENCES OF LYSOSOMAL MEMBRANE PERMEABILIZATION FOLLOWING EXPOSURE TO BIOACTIVE PARTICLES

Exposure to bioactive environmental particles and engineered nanoparticles are a significant public health concern. Inhalation of bioactive particles can result in chronic inflammation, which drives tissue remodeling and fibrosis. Furthermore, chronic inflammation can increase individual susceptibility to other diseases including cancer and autoimmune diseases. The macrophage is the critical cell in particle clearance following exposure, and is central to the inflammatory responses and tissue remodeling. Phagocytosed bioactive particles within macrophages cause cytotoxicity and activation of the NLRP3 inflammasome, outcomes that are both essential to inflammation and disease development. However, mechanisms that regulate NLRP3 inflammasome activity and cytotoxicity have not fully been elucidated. The objective of this body of work was to further define common yet critical mechanisms that cause and/or mediate NLRP3 inflammasome activity following exposure to bioactive particles. In these studies we demonstrate that bioactive particles including silica and engineered nanomaterials cause lysosome membrane permeabilization (LMP) and the release of lysosomal proteases, which precedes and facilitates NLRP3 inflammasome activation. Bioactive particles cause LMP through a mechanism that requires phagolysosome acidification. LMP and the activation of the NLRP3 inflammasome are required for secretion of pro-inflammatory cytokines and the alarmin High Mobility Group Box 1 (HMGB1). Once secreted, HMGB1 can further drive NLRP3 inflammasome activity through sterile priming, similar to the nonsterile mechanism utilized by endotoxin. A second critical pathway for regulation of the NLRP3 inflammasome was autophagy. Mice with macrophages deficient in autophagy had greater inflammation and chronic disease following silica exposure, supporting a protective anti-inflammatory role for autophagic activity. Together, these data reveal novel critical mechanisms in the regulation of NLRP3 inflammasome activity following bioactive particle exposure, and provide multiple potential therapeutic targets for the suppression of inflammation and disease

Hazard Assessment of Abraded Thermoplastic Composites Reinforced with Reduced Graphene Oxide

Graphene-related materials (GRMs) are subject to intensive investigations and considerable progress has been made in recent years in terms of safety assessment. However, limited information is available concerning the hazard potential of GRM-containing products such as graphene-reinforced composites. In the present study, we conducted a comprehensive investigation of the potential biological effects of particles released through an abrasion process from reduced graphene oxide (rGO)-reinforced composites of polyamide 6 (PA6), a widely used engineered thermoplastic polymer, in comparison to as-produced rGO. First, a panel of well-established in vitro models, representative of the immune system and possible target organs such as the lungs, the gut, and the skin, was applied. Limited responses to PA6-rGO exposure were found in the different in vitro models. Only as-produced rGO induced substantial adverse effects, in particular in macrophages. Since inhalation of airborne materials is a key occupational concern, we then sought to test whether the in vitro responses noted for these materials would translate into adverse effects in vivo. To this end, the response at 1, 7 and 28 days after a single pulmonary exposure was evaluated in mice. In agreement with the in vitro data, PA6-rGO induced a modest and transient pulmonary inflammation, resolved by day 28. In contrast, rGO induced a longer-lasting, albeit moderate inflammation that did not lead to tissue remodeling within 28 days. Taken together, the present study suggests a negligible impact on human health under acute exposure conditions of GRM fillers such as rGO when released from composites at doses expected at the workplace